Immersion oil for fluorescence microscopy

ABSTRACT

A heavy mineral oil having a relatively high viscosity is employed as an immersion oil for use in fluorescence microscopy. The oil exhibits no auto fluorescent characteristics while exhibiting a compatible refractive index to allow the same to be used with condenser/objective lens systems in fluorescence microscopy.

BACKGROUND OF THE INVENTION

Immersion oil in microscopy is usually applied as a drop connecting thespecimen (or the thin glass cover slip on top of the specimen) with theobjective lens (usually the lower lens) of the microscope. The immersionoil by replacing the air that would normally fill the space between thespecimen and lens, improves the image brightness and resolution becauseit better matches the optical requirements of refractive index andoptical dispersion (the variation of refractive index with thewavelength of light) of the optics of the microscope than does air. Airhas a refractive index of 1.000 while the ideal immersion oil has arefractive index of 1.518 (using 5461 angstrom light). Immersion oil canalso be used on the condenser lens, or between the slide and cover slip,or anywhere in the optical system of a microscope or other device whereit is compatible and useful to improve the quality of the transmittedlight.

Fluorescence is the property of a material that when illuminated bylight of one wavelength, emits (Fluoresces) light of another wavelength.In fluoresecent microscopy it is the fluorescence of the specimen thatis viewed. If the immersion oil used also fluoresces, this can causesufficient background fluorescence to obscure the fluorescence of thespecimen.

In regard to providing an immersion oil for fluorescence microscopesreference is made to U.S. Pat. No. 4,108,794 entitled OIL IMMERSION FORFLUORESCENCE MICROSCOPES issued on Aug. 22, 1978 to K. Yonekubo. Thispatent discloses an immersion oil consisting of silicone oil forfluorescence microscopes which does not emit any auto fluorescence. Inany event, the refractive index of such an oil is low as compared to thedesired index of 1.518. The oil is relatively expensive and difficult towork with.

In spite of this, there are currently no immersion oils on the marketthat have the necessary optical properties and do not fluoresce.Although much of fluorescent microscopy can be done despite thebackground fluorescence of the immersion oil used a non-fluorescingimmersion oil of the proper optical nature would be of great use incurrent techniques and might permit new techniques.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENT

An immersion oil for use in fluorescence microscopy which oil exhibitssubstantially little background fluorescence when illuminated by nearultra violet light comprising a high viscosity mineral oil having aviscosity of greater than 150 centistokes.

DETAILED DESCRIPTION OF THE INVENTION

Standard type white mineral oil exhibits little or no backgroundfluorescence when illuminated by near ultra violet light which generallycauses the most noticeable background fluorescence in immersion oil.Mineral oil is available in grades ranging from a low viscosity oil ofabout 13 centistokes and a refractive index of about 1.46 (using 5461angstrom light) to a high viscosity oil of about 160 centistokes and arefractive index of 1.48. The higher viscosity oil is not only closestto the ideal refractive index of 1.518 but the higher viscosity does abetter job of bridging the gap between the microscope specimen (or coverslip) and objective lens. The use of a mineral oil with a viscosity ofgreater than 150 centistokes will provide a proper refractive index andserve as an immersion oil for fluorescence microscopy. Mineral oil issometimes referred to as Liquid Petroleum and is a mixture of liquidhydrocarbons from petroleum. It is a colorless, oily liquid which haslittle taste and odor even when warmed. The heavy mineral oils which maybe employed as immersion oils have densities between 0.875-0.905.

EXAMPLE

    ______________________________________                                        Properties of a heavy mineral oil                                             ______________________________________                                        Refractive Index at 23° C.                                             nD (5893 A)           1.478                                                   ne (5461 A)           1.480                                                   Optical dispersion as Abbe Ve                                                                       56-57                                                   Viscosity at 25° C.                                                                          162 centistokes                                         Density at 25° C. (gm/cc)                                                                    0.88                                                    ______________________________________                                    

This immersion oil, although it has a refractive index slightly lessthan the ideal of ne=1.518, when used with a microscope in comparisonwith immersion oils that have the ideal refractive index, it showedlittle or no difference in the quality of the image.

We claim:
 1. In a process for using a fluorescent microscope forstudying materials which fluoresce in which process an immersion oil isemployed in conjunction with the objective lens the improvementtherewith of using a heavy mineral oil having a refractive index ofabout 1.480 as the immersion oil in said process, with said oil having adensity between 0.875 to 0.905 and which oil exhibits substantiallylittle background flourescence when illuminated by near ultravioletlight with said oil further having a viscosity of greater than 150centistokes at 25° C.
 2. The process according to claim 1 wherein saidoil has a density of 0.88 with a viscosity at 25° C. of 162 centistokes.